Preparation method of human trophoblast stem cells induced by human expansion potential stem cells

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method of human nourishing stem cells induced by human extended potential stem cells, which comprises the following specific steps: step 1, converting H9 human embryonic stem cells into adult extended potential stem cells on the basis of a feeder-layer-free system: (1) coating culture dishes with Matrigel, adding 1ml of Matrigel working solution into 35 dishes, and uniformly stirring; placing in an incubator at 37 DEG C overnight, and fully coating the dish bottom with the substrate for later use; according to the application, H9 human embryonic stem cells are converted into adult extended potential stem cells on the basis of a feeder-layer-free system; based on a human trophoblast stem cell culture system, feeder-layer-free human extended potential stem cells are differentiated into induced human trophoblast stem cells, and finally the induced human trophoblast stem cells are identified and induced to differentiate, so that interference of feeder-layer cells on the induced trophoblast stem cells is eliminated, and the induction efficiency of the induced trophoblast stem cells under the feeder-layer-free system is ensured.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing human trophoblast stem cells induced by human expanded potential stem cells. Background Art

[0002] Trophoblast stem cells (TSCs) have the ability to self-renew and differentiate, and can differentiate into trophoblasts with specific functions, such as extra villous trophoblasts (EVT) and syncytiotrophoblasts (STB). The cells have excellent proliferation and differentiation abilities, can simulate the development of trophoblasts in vitro, and can also be used in gene editing and drug research, providing a good model for the study of placental development and pregnancy diseases. In 2018, Japanese scientists successfully isolated human trophoblast stem cells from the blastocyst trophectoderm and the placental villi in early pregnancy. Subsequently, scientists have established a method for preparing induced human trophoblastic stem cells based on primitive human pluripotent stem cells and stimulated human pluripotent stem cells.

[0003] However, the existing human trophoblast stem cell system still has some problems. The primary isolated trophoblast stem cells need to be obtained through blastocysts or early pregnancy villi. The materials are not easy to obtain and the operation is complicated, with a low success rate. The induction of human trophoblast stem cells based on primitive human pluripotent stem cells requires the introduction of feeder cells, which interferes with subsequent applications and research. The human trophoblast stem cells obtained by induction based on excited human pluripotent stem cells have certain proliferation problems, and it is difficult to stably pass them in vitro for a long time.

[0004] Therefore, a method for preparing human expanded potential stem cells inducing human trophoblast stem cells is proposed to solve the above-mentioned problems. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing human expanded potential stem cells inducing human trophoblast stem cells, so as to solve the problems raised in the above background technology.

[0006] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a method for preparing human trophoblast stem cells induced by human expanded potential stem cells, and the specific steps of the preparation method are as follows:

[0007] Step 1: Convert H9 human embryonic stem cells into human expanded potential stem cells based on a feeder-free system:

[0008] (1) Matrigel-coated culture dishes: Add 1 ml of Matrigel working solution to a 35-well dish and place it in a 37°C incubator overnight to allow the matrix to fully coat the bottom of the dish for later use;

[0009] (2) The next day, human embryonic stem cells H9 were cultured at 1x10 4 The cells were inoculated at a density of 100 cells / mL in a Matrigel-coated 35-well dish in a medium containing mTeSR1 basal medium + 10 μM Y-27632. The cells were cultured in an incubator at 37°C and 5% CO2. After 12 hours, the culture medium was replaced with a feeder-free human expanded potential stem cell medium for induction. The medium was replaced once a day.

[0010] (3) Observe cell morphology and confluence. When the confluence reaches about 80%, you can consider subculturing. Before subculturing, prepare Matrigel-coated culture dishes, remove the old culture medium, rinse once with DPBS without Ca2+ and Mg2+, and add 1 ml of embryonic stem cell digestion solution ReSeLR to each 35-well dish. TM , and aspirate it within one minute, leaving a thin layer of dissociation reagent, incubate at 37°C for 5 minutes, then add culture medium to stop digestion, and pass the cells at a ratio of 1:5;

[0011] (4) After culturing for more than 5 passages, morphologically stable feeder-free expansion-potential stem cells can be obtained;

[0012] Step 2: Differentiate feeder-free human expanded potential stem cells into induced human trophoblast stem cells based on the human trophoblast stem cell culture system:

[0013] (1) Collagen Type IV coated culture dishes: Add 1 ml of collagen Type 4 working solution to a 35-well dish and place it in a 37°C incubator overnight to allow the matrix to fully coat the bottom of the dish for later use;

[0014] (2) Culture the morphologically stable human EPCs to a confluence of about 80%, rinse once with DPBS without Ca2+ and Mg2+, and add 1 ml of embryonic stem cell digestion solution ReSeLR to each 35-well dish. TM (Stemcell), and aspirate it within one minute, leaving a thin layer of dissociation reagent, incubate at 37°C for 5 minutes, add culture medium to stop digestion, and inoculate it into type IV collagen-coated culture dishes at a ratio of 1:10. The initial culture medium is feeder-free human expanded potential stem cell culture medium, which is replaced with human trophoblast stem cell induction medium after 12 hours, and the culture medium is replaced once a day;

[0015] (3) Observe the morphology and confluence of cells. Cells can be subcultured when the confluence is about 80%. Prepare type IV collagen-coated culture dishes before subculture. First, remove the old culture medium and rinse once with DPBS without Ca2+ and Mg2+. Add 1 ml of trophoblast stem cell digestion solution Tryple (Thermo Fisher) to each 35-well dish. Incubate at 37°C for 10 minutes, then add culture medium to stop digestion and subculture at a ratio of 1:4.

[0016] (4) Trophoblast-like clones can be observed after about 5-6 days of culture, and stable induced human trophoblast stem cells can be obtained after about 14 days of continuous induction;

[0017] (5) Cultivation, subculture, cryopreservation and resuscitation of induced human trophoblast stem cells. The induced human trophoblast stem cells need to be cultured in a type IV collagen-coated culture dish. The culture medium is the human trophoblast stem cell culture medium. After thawing or subculture, the medium is changed every 12 hours, and then every two days. After about 4-5 days of culture, the cells can be subcultured or cryopreserved. When the cell density reaches about 80%, the old culture medium is aspirated, and the cells are rinsed once with DPBS without Ca2+ and Mg2+. Then, the cells are incubated at 37°C for 10 minutes, and then the culture medium is added to stop the digestion. After centrifugation to remove the supernatant, the cells can be subcultured at a ratio of 1:4, or the supernatant is aspirated and 1m l After mixing the cell freezing solution, put it into the cell freezing tube, put it into the program freezing box and cool it in the -80℃ refrigerator overnight. It can be stored in the -80℃ refrigerator for a short time or in liquid nitrogen for a long time. For the recovery of induced human trophoblastic stem cells, prepare the culture dish coated with type IV collagen in advance, and thaw the induced human trophoblastic stem cells in a 37℃ water bath by rapid shaking until only a few ice crystals are left in the tube. In the biological safety cabinet, transfer the cell suspension to a 15ml centrifuge tube, slowly add 5 times the volume of trophoblastic stem cell culture medium, mix gently, centrifuge at 1000rpm for 5min, discard the supernatant, re-add the trophoblastic stem cell culture medium, mix well and plate;

[0018] Step 3: Identification and differentiation induction of induced human trophoblast stem cells.

[0019] Preferably, in the feeder-free system, the specific steps of the method for preparing the feeder-free expanded potential stem cell culture medium are as follows: (1) according to the instructions of mTeSR1 (Stemcell), the mTeSR1 supplemental solution is placed in a 4°C refrigerator overnight for thawing, and the thawed mTeSR1 supplemental solution (100 ml) is added to the mTeSR1 basal solution (basal) in a biological safety cabinet the next day and mixed thoroughly to obtain mTeSR1 basal culture medium, which is divided into 50 ml / tube and stored at -20°C; (2) before use, 50 ml of mTeSR1 basal culture medium is thawed in a 4°C refrigerator overnight, and 10 ng / ml leukemia inhibitory factor hLIF (Peprotech), 1 μM GSK-3 inhibitor CHIR99021 (Tocris), 2 μM (S)-(+)-dimethindene maleate (Tocris), and 2 μM minocycline hydrochloride are added in sequence in a biological safety cabinet. hydrochloride (Tocris), 1 μM WNT inhibitor IWR-1-endo (Sellect), 2 μM ROCK inhibitor Y27632 (Sellect), mix thoroughly and store at 4°C in the dark. The shelf life is 2 weeks.

[0020] Preferably, in the human trophoblast stem cell culture system, the specific steps of the method for preparing the human trophoblast stem cell induction medium are as follows: in a biological safety cabinet, take the basic culture medium DMEM / F12 (Gibco), and add 0.1mM β-mercaptoethanol 2-mercaptoethanol (Gibco), 0.2% fetal bovine serum FBS (Gibco), 0.5% penicillin / streptomycin mixture Penicillin / Streptomycin (Biyuntian), 0.3% bovine serum albumin BSA (Sigma), 1% insulin-transferrin-selenium-ethanolamine ITS-X (Gibco), 1.5μg / ml L-ascorbic acid L-ascorbic acid (Sigma), 50ng / ml epidermal growth factor EGF (Peprotech), 2μM GSK-3 inhibitor CHIR99021, 0.5μM TGF-β type I receptor inhibitor A83-01 (MCE), 1μM Src family kinase inhibitor SB431542 (MCE), 0.8 mM valproic acid VPA (MCE), and 5 μM ROCK inhibitor Y-27632 were mixed thoroughly and stored at 4°C in the dark. The shelf life is 2 weeks.

[0021] Preferably, the specific steps for preparing the Matrigel working solution are as follows: thaw 5 ml of Matrigel (Corning) at 4°C overnight, and the next day, mix the thawed Matrigel thoroughly in a biosafety cabinet and dispense it into 0.2 ml EP tubes (25 μL / tube) as a storage solution. When in use, add 25 μL of Matrigel storage solution to every 2 ml of DMEM / F12 basal culture medium and mix to obtain the working solution.

[0022] Preferably, the specific steps for preparing the type IV collagen working solution are as follows: dissolve 5 mg of Collagen Type 4 (Sigma) in 5 ml of DPBS at 4°C overnight, and dispense the completely dissolved Collagen Type 4 into 0.2 ml EP tubes (30 μL / tube) in a biosafety cabinet as a storage solution the next day. When in use, add 30 μL of Collagen Type 4 to every 6 ml of DPBS to obtain the working solution.

[0023] Preferably, the specific steps of configuring the STB differentiation medium are as follows: 0.1 mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR (Gibco), 0.5% penicillin / streptomycin mixture Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5 μM ROCK inhibitor Y-27632, and 2 μM forskolin (MCE) are added sequentially to the DMEM / F12 basal culture medium, mixed thoroughly, and stored at 4°C in the dark. The shelf life is 2 weeks.

[0024] Preferably, the specific steps of preparing the EVT differentiation medium are as follows: 0.1 mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR, 0.5% penicillin / streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5 μM ROCK inhibitor Y-27632, 7.5 μM A83-01, 100 ng / ml NRG1 (Peprotech) are added in sequence to EVT1#: DMEM / F12 basal culture medium;

[0025] In EVT2#: DMEM / F12 basal culture medium, add 0.1mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5μM ROCK inhibitor Y-27632, 7.5μM A83-01, mix thoroughly and store at 4℃ away from light. The shelf life is 2 weeks.

[0026] In EVT3#:DMEM / F12 basal culture medium, add 0.1mM β-mercaptoethanol, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5μM ROCK inhibitor Y-27632, and 7.5μM A83-01 in sequence, mix thoroughly, and store at 4°C in the dark. The shelf life is 2 weeks.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present application converts H9 human embryonic stem cells into human expanded potential stem cells based on a feeder-free system; differentiates feeder-free human expanded potential stem cells into induced human trophoblast stem cells based on a human trophoblast stem cell culture system, and finally identifies and induces differentiation of induced human trophoblast stem cells, thereby eliminating the interference of feeder layer cells on induced trophoblast stem cells and ensuring the induction efficiency of induced trophoblast stem cells in a feeder-free system. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The induction process and morphology of feeder-free expansion potential stem cells;

[0030] Figure 2 This is the induction process and morphology of induced human trophoblast stem cells;

[0031] Figure 3 This is the immunofluorescence staining of marker genes of induced human trophoblast stem cells;

[0032] Figure 4 The morphology of induced human trophoblast stem cells induced to differentiate into STB and EVT and the corresponding marker gene map detected by qRT-PCR. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] In the description of the present invention, it is necessary to understand that the terms "upper", "lower", "front", "back", "left", "right", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.

[0035] Example:

[0036] See also Figure 1-4 , the present invention provides a technical solution:

[0037] A method for preparing human trophoblast stem cells induced by human expanded potential stem cells, the specific steps of the preparation method are as follows:

[0038] Step 1: Convert H9 human embryonic stem cells into human expanded potential stem cells based on a feeder-free system:

[0039] (1) Matrigel-coated culture dishes: Add 1 ml of Matrigel working solution to a 35-well dish and place it in a 37°C incubator overnight to allow the matrix to fully coat the bottom of the dish for later use;

[0040] (2) The next day, human embryonic stem cells H9 were cultured at 1x10 4 The cells were inoculated at a density of 100 cells / mL in a Matrigel-coated 35-well dish in a medium containing mTeSR1 basal medium + 10 μM Y-27632. The cells were cultured in an incubator at 37°C and 5% CO2. After 12 hours, the culture medium was replaced with a feeder-free human expanded potential stem cell medium for induction. The medium was replaced once a day.

[0041] (3) Observe cell morphology and confluence. When the confluence reaches about 80%, you can consider subculturing. Before subculturing, prepare Matrigel-coated culture dishes, remove the old culture medium, rinse once with DPBS without Ca2+ and Mg2+, and add 1 ml of embryonic stem cell digestion solution ReSeLR to each 35-well dish. TM , and aspirate it within one minute, leaving a thin layer of dissociation reagent, incubate at 37°C for 5 minutes, then add culture medium to stop digestion, and pass the cells at a ratio of 1:5;

[0042] (4) After culturing for more than 5 passages, morphologically stable feeder-free expansion-potential stem cells can be obtained;

[0043] Step 2: Differentiate feeder-free human expanded potential stem cells into induced human trophoblast stem cells based on the human trophoblast stem cell culture system:

[0044] (1) Collagen Type IV coated culture dishes: Add 1 ml of collagen Type 4 working solution to a 35-well dish and place it in a 37°C incubator overnight to allow the matrix to fully coat the bottom of the dish for later use;

[0045] (2) Culture the morphologically stable human EPCs to a confluence of about 80%, rinse once with DPBS without Ca2+ and Mg2+, and add 1 ml of embryonic stem cell digestion solution ReSeLR to each 35-well dish. TM (Stemcell), and aspirate it within one minute, leaving a thin layer of dissociation reagent, incubate at 37°C for 5 minutes, add culture medium to stop digestion, and inoculate it into type IV collagen-coated culture dishes at a ratio of 1:10. The initial culture medium is feeder-free human expanded potential stem cell culture medium, which is replaced with human trophoblast stem cell induction medium after 12 hours, and the culture medium is replaced once a day;

[0046] (3) Observe the morphology and confluence of cells. Cells can be subcultured when the confluence is about 80%. Prepare type IV collagen-coated culture dishes before subculture. First, remove the old culture medium and rinse once with DPBS without Ca2+ and Mg2+. Add 1 ml of trophoblast stem cell digestion solution Tryple (Thermo Fisher) to each 35-well dish. Incubate at 37°C for 10 minutes, then add culture medium to stop digestion and subculture at a ratio of 1:4.

[0047] (4) Trophoblast-like clones can be observed after about 5-6 days of culture, and stable induced human trophoblast stem cells can be obtained after about 14 days of continuous induction;

[0048] (5) Cultivation, subculture, cryopreservation and resuscitation of induced human trophoblast stem cells. The induced human trophoblast stem cells need to be cultured in a type IV collagen-coated culture dish. The culture medium is the human trophoblast stem cell culture medium. After thawing or subculture, the medium is changed every 12 hours, and then every two days. After about 4-5 days of culture, the cells can be subcultured or cryopreserved. When the cell density reaches about 80%, the old culture medium is aspirated, and the cells are rinsed once with DPBS without Ca2+ and Mg2+. Then, the cells are incubated at 37°C for 10 minutes, and then the culture medium is added to stop the digestion. After centrifugation to remove the supernatant, the cells can be subcultured at a ratio of 1:4, or the supernatant is aspirated and 1m l After mixing the cell freezing solution, put it into the cell freezing tube, put it into the program freezing box and cool it in the -80℃ refrigerator overnight. It can be stored in the -80℃ refrigerator for a short time or in liquid nitrogen for a long time. For the recovery of induced human trophoblastic stem cells, prepare the culture dish coated with type IV collagen in advance, and thaw the induced human trophoblastic stem cells in a 37℃ water bath by rapid shaking until only a few ice crystals are left in the tube. In the biological safety cabinet, transfer the cell suspension to a 15ml centrifuge tube, slowly add 5 times the volume of trophoblastic stem cell culture medium, mix gently, centrifuge at 1000rpm for 5min, discard the supernatant, re-add the trophoblastic stem cell culture medium, mix well and plate;

[0049] Step 3: Identification and differentiation of induced human trophoblast stem cells.

[0050] In the feeder-free system, the specific steps of the method for preparing the feeder-free expansion potential stem cell culture medium are as follows: (1) according to the instructions of mTeSR1 (Stemcell), the mTeSR1 supplemental solution is placed in a 4°C refrigerator for overnight thawing, and the thawed mTeSR1 supplemental solution (100 ml) is added to the mTeSR1 basal solution (basal) in a biological safety cabinet the next day and mixed thoroughly to obtain the mTeSR1 basal culture medium, which is divided into 50 ml / tube and stored at -20°C; (2) before use, 50 ml of the mTeSR1 basal culture medium is thawed in a 4°C refrigerator overnight, and 10 ng / ml of leukemia inhibitory factor hLIF (Peprotech), 1 μM GSK-3 inhibitor CHIR99021 (Tocris), 2 μM (S)-(+)-dimethindene maleate (Tocris), and 2 μM minocycline hydrochloride are added in sequence in a biological safety cabinet. hydrochloride (Tocris), 1 μM WNT inhibitor IWR-1-endo (Sellect), 2 μM ROCK inhibitor Y27632 (Sellect), mix thoroughly and store at 4°C in the dark. The shelf life is 2 weeks.

[0051] In the human trophoblast stem cell culture system, the specific steps of the method for preparing the human trophoblast stem cell induction medium are as follows: in a biological safety cabinet, take the basic culture medium DMEM / F12 (Gibco), and add 0.1mM β-mercaptoethanol 2-mercaptoethanol (Gibco), 0.2% fetal bovine serum FBS (Gibco), 0.5% penicillin / streptomycin mixture Penicillin / Streptomycin (Biyuntian), 0.3% bovine serum albumin BSA (Sigma), 1% insulin-transferrin-selenium-ethanolamine ITS-X (Gibco), 1.5μg / ml L-ascorbic acid L-ascorbic acid (Sigma), 50ng / ml epidermal growth factor EGF (Peprotech), 2μM GSK-3 inhibitor CHIR99021, 0.5μM TGF-β type I receptor inhibitor A83-01 (MCE), 1μM Src family kinase inhibitor SB431542 (MCE), 0.8 mM valproic acid VPA (MCE), and 5 μM ROCK inhibitor Y-27632 were mixed thoroughly and stored at 4°C in the dark. The shelf life is 2 weeks.

[0052] The specific steps for preparing the Matrigel working solution are as follows: thaw 5 ml of Matrigel (Corning) at 4°C overnight. The next day, mix the thawed Matrigel thoroughly in a biosafety cabinet and dispense into 0.2 ml EP tubes (25 μL / tube) as storage solution. When using, add 25 μL of Matrigel storage solution to every 2 ml of DMEM / F12 basal culture medium and mix well to obtain the working solution.

[0053] The specific steps for preparing the type IV collagen working solution are as follows: dissolve 5 mg of Collagen Type 4 (Sigma) in 5 ml of DPBS at 4°C overnight, and dispense the completely dissolved Collagen Type 4 into 0.2 ml EP tubes (30 μL / tube) in a biosafety cabinet the next day as a storage solution. When used, add 30 μL of Collagen Type 4 to every 6 ml of DPBS to obtain the working solution.

[0054] The specific steps of preparing the STB differentiation medium are as follows: 0.1 mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR (Gibco), 0.5% penicillin / streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5 μM ROCK inhibitor Y-27632, and 2 μM forskolin (MCE) are added to the DMEM / F12 basal culture medium in sequence, and the mixture is stored at 4°C in the dark after being fully mixed. The shelf life is 2 weeks.

[0055] The specific steps of preparing the EVT differentiation medium are as follows: 0.1 mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5 μM ROCK inhibitor Y-27632, 7.5 μM A83-01, 100 ng / ml NRG1 (Peprotech) are added in sequence to EVT1#: DMEM / F12 basal culture medium;

[0056] In EVT2#: DMEM / F12 basal culture medium, add 0.1mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5μM ROCK inhibitor Y-27632, 7.5μM A83-01, mix thoroughly and store at 4℃ away from light. The shelf life is 2 weeks.

[0057] In EVT3#:DMEM / F12 basal culture medium, add 0.1mM β-mercaptoethanol, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5μM ROCK inhibitor Y-27632, and 7.5μM A83-01 in sequence, mix thoroughly, and store at 4°C in the dark. The shelf life is 2 weeks.

[0058] Verification Example 1:

[0059] Immunofluorescence identification of the induced trophoblastic stem cells provided in the example:

[0060] The induced trophoblastic stem cells were placed on a confocal culture dish according to the subculturing method of the embodiment, and were cultured to a confluence of about 60% for immunofluorescence staining. The specific steps are as follows:

[0061] The old culture medium was aspirated, each confocal culture dish was rinsed three times with 2 mL PBS, fixed with 1 mL 4% paraformaldehyde at room temperature for 15 min, rinsed three times with 2 ml PBS, treated with 1 ml permeabilization solution (0.3% Triton-X100) at room temperature for 30 min, rinsed three times with 2 ml PBST, added with 1 ml blocking solution (5% BSA) for 1 h at room temperature, added with 1 ml primary antibody OCT4 (Biyuntian), NANOG (Invitrogen), TP63 (Cell Signaling Technology), GATA3 (Cell Signaling Technology), respectively, treated with sealing film, incubated in a refrigerator at 4°C overnight, then discarded the primary antibody, rinsed three times with 2 ml PBST, added with 1 ml corresponding secondary antibody, incubated at room temperature for 1 h, discarded the secondary antibody, washed three times with 2 ml PBST, then added with 1 ml Hochest 33342 for staining for 10 min, discarded the staining solution, rinsed twice with PBST, observed and photographed under a confocal microscope in the dark, and recorded the results;

[0062] Verification Example 2:

[0063] The differentiation potential of the induced trophoblastic stem cells provided in the embodiment is verified, and the specific steps are as follows:

[0064] STB induced differentiation: 35 dishes were incubated with 2.5 μg / ml type IV collagen overnight in advance, and induced trophoblast stem cells were digested according to the example, and 1.0 x 10 5 The cells were seeded in 35-well dishes coated with type IV collagen. The culture medium was induced trophoblast stem cell STB differentiation medium. The medium was changed once on the third day and observation and photography were performed on the sixth day.

[0065] EVT induced differentiation: 35 dishes were incubated with 1.0 μg / ml type IV collagen overnight in advance, and 0.75×10 5 The cells were seeded in a 35-well dish coated with type IV collagen, and 2% Matrigel was added, and the culture medium was EVT1#; three days later, the culture medium was replaced with EVT2#, and 0.5% Matrigel was added. About the sixth day, TrypLE was used for digestion according to the embodiment, and the subculture ratio was 1:2. The culture medium was replaced with EVT3#, and the culture was continued for two days before observation and photography.

[0066] The induced trophoblast stem cells, STB-induced differentiation cells, and EVT-induced differentiation cells obtained in the example were rinsed once with PBS, and TRIzol lysis solution (1 mL / 35 dish) was added. After incubation at room temperature for 5 min, the cells were collected in a 1.5 mL centrifuge tube; 0.2 mL of chloroform was added to each 1 ml of TRIzol lysis solution, and the cells were incubated at room temperature for 3 min after vigorous shaking for 15 s, centrifuged at 4°C and 12000 rpm for 10 min, the supernatant aqueous phase was transferred to a new 1.5 mL centrifuge tube, 0.5 mL of isopropanol was added, and the cells were placed at room temperature for 10 min; the cells were centrifuged at 4°C and 12000 rpm for 10 min, and 1 mL of 75% ethanol was added to wash the RNA precipitate after the supernatant was removed; the cells were centrifuged at 4°C and 12000 rpm for 5 min, the supernatant was discarded, and the cells were placed to dry at room temperature; 50 μL of RNase-free water was added, and the RNA concentration was measured using NanoDrop.

[0067] The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit (PrimeScriptTM RT reagent Kit, TaKaRa, RR037A). The reverse transcription system is as follows:

[0068] 5X PrimeScript Buffer 2μL

[0069] PrimeScript RT Enzyme Mix I 0.5μL

[0070] Oligo dT Primer (50 μM) 0.5 μL

[0071] Random 6mers (100 μM) 0.5 μL

[0072] Total RNA 1μg

[0073] Total 10μL

[0074] Reaction conditions: 37℃ for 15min, 85℃ for 5s.

[0075] Then the fluorescence quantitative reagent (TB Premix Ex Taq TM , TaKaRa, RR420B) for qRT-PCR detection;

[0076] The reaction system is as follows:

[0077] TB Premix Ex Taq (2X) 12.5 μl

[0078] PCR Forward Primer(10μM)0.5μl 0.2μM

[0079] PCR Reverse Primer(10μM)0.5μl 0.2μM

[0080] DNA template (<100ng) 2μl

[0081] Total 25μL

[0082] Reaction conditions:

[0083] 95°C for 5 s, 60°C for 30 s, for a total of 40 cycles.

[0084]

[0085]

[0086] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any figure marks in the claims should not be regarded as limiting the claims involved.

[0087] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing human trophoblast stem cells induced by human expanded potential stem cells, characterized in that: The specific steps of the preparation method are as follows: Step 1: Convert H9 human embryonic stem cells into human expanded potential stem cells based on a feeder-free system: (1) Matrigel-coated culture dishes: Add 1 ml of Matrigel working solution to a 35-well dish and place it in a 37°C incubator overnight to allow the matrix to fully coat the bottom of the dish for later use; (2) The next day, human embryonic stem cells H9 were cultured at 1x10 4 The cells were inoculated at a density of 100 cells / mL in a Matrigel-coated 35-well dish in a medium containing mTeSR1 basal medium + 10 μM Y-27632. The cells were cultured in an incubator at 37°C and 5% CO2. After 12 hours, the culture medium was replaced with a feeder-free human expanded potential stem cell medium for induction. The medium was replaced once a day. (3) Observe cell morphology and confluence. When the confluence reaches about 80%, you can consider subculturing. Before subculturing, prepare Matrigel-coated culture dishes, remove the old culture medium, rinse once with DPBS without Ca2+ and Mg2+, and add 1 ml of embryonic stem cell digestion solution ReSeLR to each 35-well dish. TM , and aspirate it within one minute, leaving a thin layer of dissociation reagent, incubate at 37°C for 5 minutes, then add culture medium to stop digestion, and pass the cells at a ratio of 1:5; (4) After culturing for more than 5 passages, morphologically stable feeder-free expansion-potential stem cells can be obtained; Step 2: Differentiate feeder-free human expanded potential stem cells into induced human trophoblast stem cells based on the human trophoblast stem cell culture system: (1) Coating the culture dish with type IV collagen: Add 1 ml of collagen type 4 working solution to a 35-well dish and place it in a 37°C incubator overnight to allow the matrix to fully coat the bottom of the dish for later use; (2) Culture the morphologically stable human EPCs to a confluence of about 80%, rinse once with DPBS without Ca2+ and Mg2+, and add 1 ml of embryonic stem cell digestion solution ReSeLR to each 35-well dish. TM (Stemcell), and aspirate it within one minute, leaving a thin layer of dissociation reagent, incubate at 37°C for 5 minutes, add culture medium to stop digestion, and inoculate it into type IV collagen-coated culture dishes at a ratio of 1:

10. The initial culture medium is feeder-free human expanded potential stem cell culture medium, which is replaced with human trophoblast stem cell induction medium after 12 hours, and the culture medium is replaced once a day; (3) Observe the morphology and confluence of cells. Cells can be subcultured when the confluence is about 80%. Prepare type IV collagen-coated culture dishes before subculture. First, remove the old culture medium and rinse once with DPBS without Ca2+ and Mg2+. Add 1 ml of trophoblast stem cell digestion solution Tryple (Thermo Fisher) to each 35-well dish. Incubate at 37°C for 10 minutes, then add culture medium to stop digestion and subculture at a ratio of 1:

4. (4) Trophoblast-like clones can be observed after about 5-6 days of culture, and stable induced human trophoblast stem cells can be obtained after about 14 days of continuous induction; (5) Cultivation, subculture, cryopreservation and resuscitation of induced human trophoblast stem cells. The induced human trophoblast stem cells need to be cultured in a type IV collagen-coated culture dish. The culture medium is the human trophoblast stem cell culture medium. After thawing or subculture, the medium is changed every 12 hours, and then every two days. After about 4-5 days of culture, the cells can be subcultured or cryopreserved. When the cell density reaches about 80%, the old culture medium is aspirated, and the cells are rinsed once with DPBS without Ca2+ and Mg2+. Then, the cells are incubated at 37°C for 10 minutes, and then the culture medium is added to stop the digestion. After centrifugation to remove the supernatant, the cells can be subcultured at a ratio of 1:4, or the supernatant is aspirated and 1m l After mixing the cell freezing solution, put it into the cell freezing tube, put it into the program freezing box and cool it in the -80℃ refrigerator overnight. It can be stored in the -80℃ refrigerator for a short time or in liquid nitrogen for a long time. For the recovery of induced human trophoblastic stem cells, prepare the culture dish coated with type IV collagen in advance, and thaw the induced human trophoblastic stem cells in a 37℃ water bath by rapid shaking until only a few ice crystals are left in the tube. In the biological safety cabinet, transfer the cell suspension to a 15ml centrifuge tube, slowly add 5 times the volume of trophoblastic stem cell culture medium, mix gently, centrifuge at 1000rpm for 5min, discard the supernatant, re-add the trophoblastic stem cell culture medium, mix well and plate; Step 3: Identification and differentiation induction of induced human trophoblast stem cells.

2. The method for preparing human trophoblast stem cells induced by human expanded potential stem cells according to claim 1, characterized in that: In the feeder-free system, the specific steps of the method for preparing the feeder-free expansion potential stem cell culture medium are as follows: (1) according to the instructions of mTeSR1 (Stemcell), the mTeSR1 supplemental solution is placed in a 4°C refrigerator for overnight thawing, and the thawed mTeSR1 supplemental solution (100 ml) is added to the mTeSR1 basal solution (basal) in a biological safety cabinet the next day and mixed thoroughly to obtain the mTeSR1 basal culture medium, which is divided into 50 ml / tube and stored at -20°C; (2) before use, 50 ml of the mTeSR1 basal culture medium is thawed in a 4°C refrigerator overnight, and 10 ng / ml of leukemia inhibitory factor hLIF (Peprotech), 1 μM GSK-3 inhibitor CHIR99021 (Tocris), 2 μM (S)-(+)-dimethindene maleate (Tocris), and 2 μM minocycline hydrochloride are added in sequence in a biological safety cabinet. hydrochloride (Tocris), 1 μM WNT inhibitor IWR-1-endo (Sellect), 2 μM ROCK inhibitor Y27632 (Sellect), mix thoroughly and store at 4°C in the dark. The shelf life is 2 weeks.

3. The method for preparing human trophoblast stem cells induced by human expanded potential stem cells according to claim 1, characterized in that: In the human trophoblast stem cell culture system, the specific steps of the method for preparing the human trophoblast stem cell induction medium are as follows: in a biological safety cabinet, take the basic culture medium DMEM / F12 (Gibco), and add 0.1mM β-mercaptoethanol 2-mercaptoethanol (Gibco), 0.2% fetal bovine serum FBS (Gibco), 0.5% penicillin / streptomycin mixture Penicillin / Streptomycin (Biyuntian), 0.3% bovine serum albumin BSA (Sigma), 1% insulin-transferrin-selenium-ethanolamine ITS-X (Gibco), 1.5μg / ml L-ascorbic acid L-ascorbic acid (Sigma), 50ng / ml epidermal growth factor EGF (Peprotech), 2μM GSK-3 inhibitor CHIR99021, 0.5μM TGF-β type I receptor inhibitor A83-01 (MCE), 1μM Src family kinase inhibitor SB431542 (MCE), 0.8 mM valproic acid VPA (MCE), and 5 μM ROCK inhibitor Y-27632 were mixed thoroughly and stored at 4°C in the dark. The shelf life is 2 weeks.

4. The method for preparing human trophoblast stem cells induced by human expanded potential stem cells according to claim 1, characterized in that: The specific steps for preparing the Matrigel working solution are as follows: thaw 5 ml of Matrigel (Corning) at 4°C overnight. The next day, mix the thawed Matrigel thoroughly in a biosafety cabinet and dispense into 0.2 ml EP tubes (25 μL / tube) as storage solution. When using, add 25 μL of Matrigel storage solution to every 2 ml of DMEM / F12 basal culture medium and mix well to obtain the working solution.

5. The method for preparing human trophoblast stem cells induced by human expanded potential stem cells according to claim 1, characterized in that: The specific steps for preparing the type IV collagen working solution are as follows: dissolve 5 mg of Collagen Type 4 (Sigma) in 5 ml of DPBS at 4°C overnight, and dispense the completely dissolved Collagen Type 4 into 0.2 ml EP tubes (30 μL / tube) in a biosafety cabinet as a storage solution the next day. When used, add 30 μL of Collagen Type 4 to every 6 ml of DPBS to obtain the working solution.

6. The method for preparing human trophoblast stem cells induced by human expanded potential stem cells according to claim 1, characterized in that: The specific steps of preparing the STB differentiation medium are as follows: 0.1 mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR (Gibco), 0.5% penicillin / streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5 μM ROCK inhibitor Y-27632, and 2 μM forskolin (MCE) are added to the DMEM / F12 basal culture medium in sequence, and the mixture is stored at 4°C in the dark after being fully mixed. The shelf life is 2 weeks.

7. The method for preparing human trophoblast stem cells induced by human expanded potential stem cells according to claim 1, characterized in that: The specific steps of preparing the EVT differentiation medium are as follows: 0.1 mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5 μM ROCK inhibitor Y-27632, 7.5 μM A83-01, 100 ng / ml NRG1 (Peprotech) are added in sequence to EVT1#: DMEM / F12 basal culture medium; In EVT2#: DMEM / F12 basal culture medium, add 0.1mM β-mercaptoethanol 2-mercaptoethanol, 4% serum substitute KSR, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5μM ROCK inhibitor Y-27632, 7.5μM A83-01, mix thoroughly and store at 4℃ away from light. The shelf life is 2 weeks. In EVT3#:DMEM / F12 basal culture medium, add 0.1mM β-mercaptoethanol, 0.5% Penicillin / Streptomycin, 0.3% bovine serum albumin BSA, 1% insulin-transferrin-selenium-ethanolamine ITS-X, 2.5μM ROCK inhibitor Y-27632, and 7.5μM A83-01 in sequence, mix thoroughly, and store at 4°C in the dark. The shelf life is 2 weeks.

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